Genetic and transcriptomic approaches to understanding and engineering terpenoid biosynthesis in Mentha × piperita

Holland, Alistair 2025. Genetic and transcriptomic approaches to understanding and engineering terpenoid biosynthesis in Mentha × piperita. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Mentha × piperita cv. Black Mitcham is an economically important aromatic crop valued for its essential oil, particularly the monoterpenoid menthol. This thesis focuses on developing experimental platforms to investigate and engineer the menthol biosynthetic pathway. An efficient in vitro regeneration protocol utilising 1-naphthaleneacetic acid and thidiazuron was established, enabling rapid callus formation and de novo shoot induction. Three transformation strategies were explored, with biolistic delivery of exogenous DNA and direct injection of Agrobacterium tumefaciens yielding the most consistent transgene expression. Heterologous reconstitution of menthol biosynthesis was attempted in Nicotiana benthamiana and Arabidopsis thaliana. Transient and stable expression of Mentha x piperita GERANYL DIPHOSPHATE SYNTHASE SMALL SUBUNIT, Picea abies LIMONENE SYNTHASE, and Mentha x piperita LIMONENE-3-HYDROXYLASE in Nicotiana benthamiana enabled production of limonene and (–)-trans-isopiperitenol, while stable transformation in Arabidopsis thaliana generated additional intermediates. Additionally, increasing the supply of terpenoid precursors by co-expression of Nicotiana tabacum 1-DEOXY-D-XYLULOSE 5-PHOSPHATE SYNTHASE increased precursor availability, enhancing limonene production. Essential oil profiling revealed dynamic changes across development, with menthone and menthol displaying reciprocal changes between vegetative and reproductive growth phases. RNA-seq transcriptomic analysis was performed to link these compositional changes to gene expression. This transcriptomic survey provided a genome-wide view of developmental regulation, identifying developmental stage-specific expression patterns of key pathway genes. Clustering and promoter motif analysis further suggested candidate transcription factors that may regulate menthol biosynthesis, while additional genes encoding putative biosynthetic enzymes were uncovered. Together, this work delivered novel regeneration and transformation strategies for peppermint, established heterologous model systems for menthol biosynthesis pathway intermediate biosynthesis, and provided regulatory insights from transcriptomics, laying the foundation for metabolic engineering of menthol in Mentha x piperita cv. Black Mitcham and alternative plant hosts.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Schools > Biosciences
Subjects:	Q Science > Q Science (General)
Date of First Compliant Deposit:	20 March 2026
Last Modified:	20 Mar 2026 17:03
URI:	https://orca.cardiff.ac.uk/id/eprint/185904

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